Many of the prior art methods of preparing tissue specimens for histology use incubation in separate solutions of phosphate-buffered 10% formalin for fixation, a series of increasing concentrations of ethanol, and/or isopropanol for dehydration, and xylene for clearing tissue of dehydration agent, prior to impregnation. Because of the time required for this process, usually 8 hours or longer, it is customary to complete these separate steps—fixation, dehydration, clearing, and impregnation—overnight in automated mechanical instruments designed for those tasks.
The most rapid tissue preservation methods of the prior art rely on microwave processing which does not maintain the non-aqueous character of the reagents that is required in order to prevent burning of the tissue specimens. Moreover, prior fixation processes are plagued by irreversible damage (e.g., hydrolysis of a phosphodiester bond and/or deamidation) to the structure of nucleic acids (e.g., DNA, and especially RNA) that limits the application of genetic techniques for diagnosis and research. Consequently, most DNA and certainly RNA analysis require special precautions with the handling of material, such as immediate freezing of fresh tissues to prevent degradation that can impair retrospective genetic analysis.
Most microwave reagents have poor diffusivity and will not penetrate the specimen properly without pretreatment. Specimens must be sectioned to about 1.5 mm or less, and most preferably to about 1 mm or less, for the process to work. Specimens also require treatment for 30 minutes or more in a pre-processing solution to harden the specimen, so that it can be sectioned to the required thickness.
In some prior processing devices, when the reagents are evacuated after reacting with the specimens, scale, slime and deposits are formed. Scale forms where it can be tolerated least-on heat transfer surfaces. It is in this location that the conditions necessary to cause the precipitation of salts are found. At areas of heat transfer and low flow rates, there is a significant increase in the dissolved solids concentration. There is also a localized temperature rise. The crystallization of scale on these surfaces is a slow process. This promotes the formation of a fairly well defined crystal growth, especially considering the varying composition present in the used fixative. Slow, in-place crystal growth forms a hard, dense, glassy and highly insulating material that is deposited on the evacuation valves and associated conduit surfaces. Some forms of scale are so tenacious that they resist any type of removal, mechanical or chemical. Scales forming on the moving parts of valves stick together and require replacement of the valves and valve motors.
There are also problems associated with impeded work flow in the pathology laboratory necessitated by the requisite batch processing of specimens, the safety concerns that attend having instruments operating overnight, the risk of possible instrument failures, the need to monitor the instruments, and the waste of using large volumes of reagents for such processing when automated.
Expensive measures are required to prevent exposure of laboratory personnel to noxious fumes and toxic substances associated with the reagents used in this process. Also, the large volumes of reagent waste and Paraffin debris produced by the conventional methodology will pollute the environment if not properly disposed.
Prior art processing of tissue is accomplished in a plurality of reactors requiring manual or robotic handling and transfer of the specimens.
Most significantly, the prior art fails to teach an efficient solvent to remove cellular solutes in a single step.
A typical tissue cell contains: 19.44% ether-extractable material (lipids), 55.13% moisture 18.62% protein, and 5.43% ash. The objective is to remove the lipids and free water and replace them with a preserving compound such as Paraffin. Fixation is the first step in preparing cell and tissue specimens for use in a wide range of clinical and analytical testing. A good fixative should harden cell and tissue components. The chemical process of tissue modification by a fixative is gradual and complex, involving penetration into the tissue and a variety of chemical reactions. Formaldehyde fixatives (formalin) are very reactive electrophilic reagents that fix tissue by covalent bonding to reactive functional groups present in tissue. Neutral Buffered Formalin (NBF) is the most widely used fixative for preserving cell and tissue specimens. It is relatively inexpensive, simple to use, and provides consistent results. NBF developed by the US Armed Forces Institute of Pathology is the exclusive aqueous fixative used in automated tissue processors. The formulation is a 1:10 v/v dilution of 37% w/v formaldehyde in a phosphate buffer.
However, when tissue specimens are processed at an elevated temperature (35-50 degrees C.) for a long period of time, e.g., 2 to 12 hours, solid materials tend to accumulate in the processor. Frequent maintenance is required to prevent instrument failure. Several processing device components can be affected by NBF. They include reservoir connections, rotary valve, retort sensors, overflow sensors, solenoid valves, pressure/vacuum regulator manifolds, process pumps and associated plumbing.